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21 January 2002, Volume 21, Number 4, Pages 598-610
Table of contents    Previous  Article  Next   [PDF]
Review
Alternative lengthening of telomeres in mammalian cells
Jeremy D Henson, Axel A Neumann, Thomas R Yeager and Roger R Reddel

Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney 2145, Australia

Correspondence to: R R Reddel, Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney 2145, Australia. E-mail: rreddel@cmri.usyd.edu.au

Abstract

Some immortalized mammalian cell lines and tumors maintain or increase the overall length of their telomeres in the absence of telomerase activity by one or more mechanisms referred to as alternative lengthening of telomeres (ALT). Characteristics of human ALT cells include great heterogeneity of telomere size (ranging from undetectable to abnormally long) within individual cells, and ALT-associated PML bodies (APBs) that contain extrachromosomal telomeric DNA, telomere-specific binding proteins, and proteins involved in DNA recombination and replication. Activation of ALT during immortalization involves recessive mutations in genes that are as yet unidentified. Repressors of ALT activity are present in normal cells and some telomerase-positive cells. Telomere length dynamics in ALT cells suggest a recombinational mechanism. Inter-telomeric copying occurs, consistent with a mechanism in which single-stranded DNA at one telomere terminus invades another telomere and uses it as a copy template resulting in net increase in telomeric sequence. It is possible that t-loops, linear and/or circular extrachromosomal telomeric DNA, and the proteins found in APBs, may be involved in the mechanism. ALT and telomerase activity can co-exist within cultured cells, and within tumors. The existence of ALT adds some complexity to proposed uses of telomere-related parameters in cancer diagnosis and prognosis, and poses challenges for the design of anticancer therapeutics designed to inhibit telomere maintenance.

Oncogene (2002) 21, 598-610 DOI: 10.1038/sj/onc/1205058

Keywords

telomere; alternative lengthening of telomeres; ALT-associated PML bodies; recombination; immortalization; cancer

Abbreviations

ALT, alternative lengthening of telomeres; APB, ALT-associated PML body; DSB, double-strand break; ECTR, extrachromosomal telomeric repeats; FISH, fluorescent in situ hybridization; HPV, human papillioma virus; HR, homologous recombination; LFS, Li-Fraumeni syndrome; PNB, PML nuclear body; PD, population doubling; SV40, simion virus 40; TRD, telomeric rapid deletion; TERT, telomerase reverse transcriptase; TR, telomerase RNA; TRF, terminal restriction fragment.

Some mammalian cells without any telomerase activity are able to maintain the length of their telomeres for many population doublings (PDs) (Bryan et al., 1995; Hande et al., 1999; Niida et al., 2000; Rogan et al., 1995), thus indicating the existence of one or more non-telomerase mechanisms for telomere maintenance that have been termed Alternative Lengthening of Telomeres (ALT) (Bryan and Reddel, 1997). To date, clear evidence for ALT activity has only been found in abnormal situations, including human tumors, immortalized human cell lines (Table 1), and in telomerase-null mouse cell lines (Bryan et al., 1995, 1997a; Hande et al., 1999; Niida et al., 2000). There is also suggestive evidence for ALT activity in the tissues of late generation telomerase-null mice (Hande et al., 1999; Herrera et al., 2000). It seems likely that understanding this form of telomere maintenance will have important implications for the diagnosis and treatment of cancer. Here we review what is known about ALT in mammalian (primarily human) cells, and discuss proteins that may be involved in these processes.

Telomere length phenotype of ALT cells

ALT cells have a characteristic heterogeneous telomere length phenotype. Telomeres are normally maintained in the human germline at lengths around 15 kb (Allshire et al., 1989; de Lange et al., 1990), as measured by terminal restriction fragment (TRF) Southern analysis. TRFs include up to 5 kb of non-telomere repeat sequence (Henderson et al., 1996 and references therein). For normal somatic cells in vitro, the TRF length progressively declines at a rate of 40-200 base pairs (bp) per cell division to 5-8 kb at senescence (Harley, 1997; Martens et al., 2000; Wright et al., 1997). In most telomerase-positive human cancers or immortal cell lines, TRF lengths are relatively homogeneous with the mean length usually less than 10 kb (Bryan et al., 1995; de Lange, 1995; Park et al., 1998). In contrast, all of the human ALT+ cell lines and cancers analysed so far have a longer mean TRF length with a very wide length distribution (Figure 1): the mean is around 20 kb, and TRF lengths range from less than 3 kb to over 50 kb (Bryan et al., 1995, 1997a; Grobelny et al., 2000; Murnane et al., 1994; Opitz et al., 2001). Consequently, pulsed field rather than conventional gel electrophoresis is preferable for analysing TRFs of ALT cells. In cells that become immortalized and activate ALT during culturing in vitro, there is a good temporal correlation between the immortalization event and the occurrence of the characteristic ALT telomere length phenotype (Figure 1) (Yeager et al., 1999). Visualization of telomeres by fluorescence in situ hybridization (FISH) shows that the telomere length heterogeneity characteristic of ALT cell populations reflects the heterogeneity that exists within individual cells (Figure 2a). Some chromosome ends have no detectable telomeric sequence while others within the same cell have very strong telomere signals (Lansdorp et al., 1997; Perrem et al., 2001).

ALT-associated PML bodies

Another characteristic of all human ALT cell lines examined to date is the presence of nuclear structures referred to as ALT-associated PML bodies (APBs), i.e., PML nuclear bodies (PNBs) with ALT-specific contents. PNBs are aggregates of PML and other proteins that are usually bound to the nuclear matrix. PNBs are the subject of many recent articles that are too numerous to cite here; the reader is referred to some of the many recent reviews (e.g., Hodges et al., 1998; Maul et al., 2000; Ruggero et al., 2000; Zhong et al., 2000). PNBs are present in many but not all tissues. They are dynamic structures with PML and other proteins continually being incorporated and released. Their number, size, morphology, constituents and function may be influenced by the expression, alternative splicing and post-translational modification of PML and may vary with the cell cycle, state of the cell and external influences. Some other proteins are also important for the formation of PNBs, but many components may only be present in specific cellular contexts and only in a subset of PNBs. The processes in which PNBs are claimed to be involved include tumor suppression, cell cycle regulation, senescence, apoptosis, immune and inflammatory responses, antigen presentation, protein refolding and degradation, and differentiation. PML and other common constituents of PNBs may regulate transcription and modify chromatin. PNBs are closely associated with replication domains and interaction of viral DNA with PNBs may be necessary for optimal viral replication. It has been proposed that PNBs facilitate this vast array of functions by sequestering and releasing proteins, localizing proteins to sites of action and facilitating interactions between other proteins including those that result in post-translational modifications.

APBs are distinguished from other PNBs by their contents, including telomeric DNA and the telomere binding proteins, TRF1 and TRF2 (Yeager et al., 1999). APBs have also been found to contain a range of proteins involved in DNA recombination and replication: RAD51, RAD52, RPA, MRE11, RAD50, NBS1, BLM and WRN (Table 2) (Johnson et al., 2001; Wu et al., 2000; Yankiwski et al., 2000; Yeager et al., 1999; Zhu et al., 2000). Like their normal counterparts, APBs have the appearance of disc or ring shaped structures in two dimensions, often with PML detected in the outer rim (Figure 3). In view of the postulated functions for PNBs, it is possible that APBs may focus, colocalize, or modify proteins required for the ALT mechanism. It is also possible that APBs are involved in removing by-products of the ALT process, as there is some evidence that PNBs could be sites of intranuclear proteolysis (Lallemand-Breitenbach et al., 2001). Although all ALT cell lines examined so far have APBs, they are seen in only about 5% of interphase cells within an exponentially dividing ALT+ population (Yeager et al., 1999). Most cells with APBs are in the late S/G2/M phase of the cell cycle (Grobelny et al., 2000; Wu et al., 2000).

Prior to the observation that telomeric DNA is present in APBs (Yeager et al., 1999), DNA had not been found in PNBs. It has recently been shown that PML may colocalize in nuclear foci with BLM, RPA and RAD51 in response to DNA damage (Bischof et al., 2001). Although the relationship between PNBs and RAD51 foci is not clear, it seems possible that the function of APBs is to repair telomeric DNA that is recognized by the cell as being damaged.

The telomeric DNA in APBs may be a subset of the total extrachromosomal telomeric repeats (ECTR) that have been detected in various types of cells (Ogino et al., 1998; Tokutake et al., 1998). In general, ECTR are not detectable in telomerase-positive immortalized cells or in normal human cells (Ogino et al., 1998). However, ECTR have been reported in mortal EBV-transformed B lymphoblastoid cell lines (Sugimoto et al., 1999). They may also be present in otherwise normal fibroblasts from individuals with ataxia telangiectasia (Hande et al., 2001), a condition associated with accelerated telomere shortening in vitro (Metcalfe et al., 1996). In contrast, there seems to be a tight correlation between the presence of APBs within a cell line and the presence of ALT, as manifested by the characteristic telomere length pattern. APBs have been found in 17/17 ALT+ and 0/20 telomerase-positive cell lines, and in 0/5 mortal cell strains (Yeager et al., 1999 and T Yeager et al., unpublished data). APBs can be detected in ALT+ tumors (Yeager et al., 1999), although the number examined so far is still small. There is a temporal correlation between the immortalization event and the occurrence of APBs (Yeager et al., 1999). Conversely, when ALT is repressed in somatic cell hybrids, APBs eventually disappear (Perrem et al., 2001).

ALT genetics and repression

Immortalization usually depends on recessive mutations (Pereira-Smith and Smith, 1983), and human cell lines have been assigned to at least four complementation groups for immortality (Pereira-Smith and Smith, 1988). Presumably, at least some of the genes corresponding to these complementation groups are repressors of a telomere maintenance mechanism: telomerase or ALT. However, some of these genes may act through other pathways because the mortal phenotype can be restored in somatic cell hybrids despite the presence of telomerase activity (as detected by an in vitro assay, which does not necessarily reflect continuing telomerase activity at the telomere) (Bryan et al., 1995). That ALT results from recessive mutation(s) was demonstrated by the observation that fusion of an ALT+ immortal cell line with normal cells resulted in senescent hybrids that had lost the ALT telomere phenotype (Perrem et al., 1999). Further, some telomerase-positive immortalized cells appear to contain repressors of ALT activity: ALT was repressed in immortal hybrid cells formed by fusing an ALT+ cell line from immortalization complementation group A with either of two telomerase-positive cell lines from the same complementation group (Perrem et al., 1999, 2001). In contrast, fusion of ALT+ and telomerase-positive cell lines from immortalization complementation group D resulted in immortal hybrids with ALT active and telomerase repressed (Katoh et al., 1998), indicating that some ALT cells may contain a repressor of telomerase.

The observation that ALT cell lines have been assigned to at least two immortalization complementation groups (A and D) suggests the possibility that there may be more than one gene that can repress ALT (Whitaker et al., 1995). Introduction of chromosome 7 into group D ALT+ cells suppressed both immortality and ALT (Nakabayashi et al., 1997; Ogata et al., 1993). The chromosome 7 gene(s) may not specifically repress ALT, however, because chromosome 7 also restored mortality to a group D telomerase-positive cell line (Ogata et al., 1995). Understanding the mechanisms whereby ALT activity is repressed in normal cells may make it possible to design anti-cancer therapies that restore this repression.

Telomerase components in ALT cell lines

The absence of telomerase activity from ALT cells correlates with lack of expression of hTERT (the telomerase catalytic subunit; TElomerase Reverse Transcriptase) and sometimes hTR (the RNA template moiety; Telomerase RNA) as well. ALT cells have undetectable levels of the full-length hTERT transcript (Kilian et al., 1997). This is associated with methylation of the hTERT CpG island, in contrast to telomerase-negative normal cells in which the CpG island is unmethylated (Dessain et al., 2000). The observation that some ALT cell lines do not express detectable levels of hTR provided definitive evidence that telomere maintenance in these lines is independent of telomerase activity (Bryan et al., 1997b). Lack of hTR expression in ALT lines is associated with promoter methylation in the gene, hTERC, that encodes it (Hoare et al., 2001). Expression of exogenous hTR in these cells did not result in telomerase activity (Bryan et al., 1997b). In ALT cells that express the hTERC gene, the sequence is wild-type (Bryan et al., 1997b).

Expression of exogenous hTERT in hTR-expressing ALT cells induces telomerase activity, as detected by an in vitro assay (Wen et al., 1998), indicating that the other telomerase subunits are expressed at sufficient levels to support telomerase activity in these cells. For hTR-negative ALT cells, expression of both exogenous hTR and hTERT was required to induce telomerase activity (Wen et al., 1998). These findings have permitted studies of the effects of telomerase (Cerone et al., 2001; Ford et al., 2001; Grobelny et al., 2001; Perrem et al., 2001) and of mutant hTR template sequence (Guiducci et al., 2001) on ALT cells.

Ability of ALT and telomerase activity to co-exist in human cells

Expression of exogenous telomerase in ALT cells is usually compatible with continued ALT activity, even though the telomerase activity lengthens the shortest telomeres (Cerone et al., 2001; Ford et al., 2001; Grobelny et al., 2001; Perrem et al., 2001). Subclones of late passage (>100 PD) ALT cells expressing telomerase activity showed >100-fold reduction in the number of chromosome ends with telomere sequences that were undetectable by FISH (Perrem et al., 2001). The very long telomeres persisted, however, and there was no significant change in the proportion of cells with APBs (Cerone et al., 2001; Grobelny et al., 2001; Perrem et al., 2001). Telomere length heterogeneity was still being generated rapidly after more than 100 PDs with telomerase activity (Perrem et al., 2001). This means that the repression of ALT seen in some ALT+´telomerase-positive hybrids is unlikely to be due to telomerase activity per se. It also suggests that ALT can act on telomeres that are not critically short, unlike the situation in yeast where telomeric recombination in telomerase-null survivors is repressed by re-expression of telomerase activity (Teng and Zakian, 1999). In one study, however, expression of telomerase in a human ALT cell line resulted in reduced evidence of ALT activity in two of nine clones (Ford et al., 2001). One possible explanation might be that ALT may be switched off as a stochastic event in some cells (as has been demonstrated for telomerase in telomerase-positive cells (Bryan et al., 1998)) which do not therefore lose proliferative capacity when exogenous telomerase is present. Another explanation could be that ALT and telomerase may compete for common molecular components or access to the telomere, and that ALT can be repressed under circumstances where a particularly high level of a telomerase subunit such as TERT is present. A further explanation might be that there is a minority of ALT cells in which telomere lengthening events only occur when telomeres become shorter than a critical length which happens to be less than that to which the exogenous telomerase activity lengthens the shortest telomeres.

Telomere dynamics in ALT consistent with recombination

The telomere length distribution in ALT cells is dynamic, with fluctuations in length occurring on individual telomeres during cellular proliferation. In a key study, Murnane and colleagues observed the following length dynamics of a tagged telomere in a telomerase-negative human cell line (Murnane et al., 1994). Telomeres underwent gradual shortening at a rate of 30-50 bp per cell division, which is similar to cells without a telomere maintenance mechanism (Martens et al., 2000). In some cells, this erosion continued until there were less than 200 bp of telomeric repeats left (without extending into the subtelomeric region) before a rapid and heterogeneous increase in length, sometimes of >23 kb, occurred. In other cells, rapid increases in telomere length occurred in telomeres that did not appear to be critically short. Rapid deletion events occurred occasionally in telomeres of any length. The frequency of these changes varied greatly between different subclones. The frequency of chromosomal fusion events seemed to be proportional to the frequency of rapid length changes. These length dynamics were all consistent with the alterations of telomere length being mediated by recombinational events (Murnane et al., 1994).

Fluctuation of telomere length within ALT cells has also been found in a subsequent study. In an ALT cell line that contained a single Y chromosome, it was shown that the Y p- and q-arm telomere length ratios varied by more than 100-fold within the cell population, in contrast to a comparable telomerase-positive cell line where the ratio varied by less than twofold (Perrem et al., 2001).

Telomeric recombination in ALT+ cells

In addition to telomere length dynamics in the telomerase-negative human cells (Murnane et al., 1994; Perrem et al., 2001), data from other organisms also suggested that telomeres may under some circumstances be maintained by a recombinational process. Recombination is the primary mechanism of telomere maintenance in the mosquito malarial vector, Anopheles gambiae (Roth et al., 1997), and possibly for the telomeres of linear mitochondrial DNA in some species of yeast (Nosek et al., 1998). Recombination is also used by some species of yeast as a back-up mechanism for telomere maintenance. In the yeast, S. cerevisiae, inactivation of telomerase leads to loss of telomeric repeats with cell division, and eventually death of most of the cells; survivors are dependent on the Rad52 gene which encodes a protein required for recombination (Lundblad and Blackburn, 1993). There are two categories of such survivors: in type I there is amplification of a subtelomeric tract repeat element with a short terminal telomeric repeat and in type II there is elongation of telomeric repeats (Le et al., 1999; Teng et al., 2000; Teng and Zakian, 1999). The telomere length phenotypes of the Type II telomerase-null survivors in Saccharomyces cerevisiae and also the Rad52-dependent telomerase-null survivors in K. lactis resemble those of ALT+ human cells (McEachern and Blackburn, 1996; Teng and Zakian, 1999). In Drosophila and some related Dipteran species, retrotransposons are utilized for telomere maintenance (Biessmann et al., 1990), but the increased TTAGGG-hybridizing DNA observed in the telomeres of ALT cells (Bryan et al., 1995) makes it unlikely that retrotransposition contributes significantly to telomere maintenance in these cells. On the basis of some of these considerations, a telomere maintenance mechanism involving inter-telomeric recombination was proposed for mammalian ALT cells (Reddel et al., 1997) (Figure 5).

Inter-telomeric recombination

Evidence for inter-telomeric recombinational events in human ALT cells was obtained by targeting a DNA tag into telomeres (Dunham et al., 2000). FISH analysis of clonal cultures showed a progressive increase in the number of tagged telomeres with increasing PDs. At PD 23 the tag was found in two or three telomeres. By PD 63 it was found on up to five telomeres in any one cell and, within the clonal population, ten different chromosomes were tagged. This phenomenon was not seen when the tag was located immediately centromeric to the telomere in ALT cells, and was not seen in telomerase-positive cells. Furthermore, chromosome specific sub-telomeric probes also showed that the increased telomeric recombination in the ALT cell line did not extend to the sub-telomeric region (Figure 4). These data are consistent with a mechanism in which the single-stranded DNA at the end of one telomere invades double-stranded DNA of another telomere and uses it as a copy template resulting in a net increase in telomeric DNA within the cell. This assay was set up to detect inter-telomeric copying of DNA sequence, and the data do not exclude the possibility that intra-telomeric strand invasion and t-loop formation (Griffith et al., 1999) also permits a telomere to elongate by using itself as a copy template, or that copying of DNA from one telomere to another has an intermediate step involving extrachromosomal telomeric DNA sequences (Dunham et al., 2000).

T-loops

It has been shown by electron microscopy that human and mouse telomeres can form loop structures, termed t-loops. The putative structure of t-loops is shown in Figure 5, and involves a single-stranded 3' overhang invading proximal duplex telomeric DNA, causing a displacement (D)-loop that is 75 to 200 nucleotides long (Griffith et al., 1999). The formation of t-loops in vitro is dependent on TRF2 and a 3' overhang; TRF2 binds near the proximal D-loop junction and is thought to be important in stabilizing the D-loop. TRF1 may help fold the t-loop (Griffith et al., 1999). Telomeres may also form loops in trypanosomes and hypotrichous ciliates (Lipps et al., 1998; Muñoz-Jordán et al., 2001; Murti and Prescott, 1999). T-loops have been postulated to be a mechanism for hiding the telomere ends from various proteins, but also provide a structure that could result in elongation or shortening of the telomere. The 3' overhang strand invasion in t-loops is equivalent locally to the structure used in recombination dependent replication. It has been suggested that replication is normally inhibited by the telomeric DNA end binding protein, POT1 (Baumann and Cech, 2001). If replication does occur on the invading strand, branch migration of the invading strand together with lagging strand synthesis may allow the t-loop to roll with replication continuing indefinitely.

T-loops may also facilitate telomeric shortening if a cross over event occurs. This could account for the rapid reduction in telomere length seen in ALT cells (Murnane et al., 1994), and in hybrid cells in which ALT is repressed (Perrem et al., 1999, 2001). A process of telomere shortening found in yeast, telomeric rapid deletion (TRD), is postulated to use a t-loop structure (Bucholc et al., 2001). TRD reduces the longer telomeres to the length of the majority of telomeres in a single cell division (Li and Lustig, 1996), and involves Rad52, Rad50 and Mre11, genes that are required for the type II the telomerase-null survivor pathway. TRD is stimulated by the hyper-recombination mutant hpr1.

T-loops could contribute to telomere lengthening in ALT cells in several ways in addition to enabling a telomere to use itself as a copy template (Figure 5). Loop-mediated excision of telomeric DNA could generate linear and maybe circular DNA that could participate in lengthening of other telomeres by rolling circle and other mechanisms described below. This could account, at least in part, for the intertelomeric copying of DNA sequences that has been observed in ALT cells (Dunham et al., 2000).

Rolling circle

Another possible recombination-mediated mechanism of telomere lengthening involves a rolling circle of replication in which the 3' single-stranded telomeric overhang invades a circle of ECTR DNA (Figure 5). Branch migration of the 3' overhang then allows rolling of the circle and essentially unlimited elongation. Artificial circular DNA containing telomeric repeats has been shown to be utilized by K. lactis to greatly extend its telomeres (McEachern, 2001). In Candida parapsilosis and other yeast species with linear mitochondrial DNA, rolling circles may be used for maintaining the mitochrondrial DNA termini (Tomaska et al., 2000). Telomere repeat circles have been found in human tumors and in a human immortal cell line (Regev et al., 1998). Other types of small circular DNA have been found in human cells and are considered to be either a marker or an enhancer of genomic instability (Cohen et al., 1997; Wahl, 1989). In yeast sgs1 mutations increase the formation of rDNA circles and possibly subtelomeric repeat circles (Sinclair and Guarente, 1997). The presence of circles containing telomere repeat DNA has not yet been documented in a human ALT line, and indeed one study found only linear ECTR in an ALT line (Ogino et al., 1998). Nevertheless, the possibility cannot yet be excluded that circular telomeric DNA sequences, perhaps generated from t-loops or stalled replication forks, could provide the substrate for rolling circle elongation of telomeres in ALT cells.

Linear ECTR DNA

ECTR DNA has been found in APBs of all ALT cell lines tested (Yeager et al., 1999), and DNA within APBs has free ends indicating that at least some of it is linear (T Yeager et al., unpublished data). Low molecular weight telomeric DNA that appears to be linear can be extracted from ALT cells (Ogino et al., 1998). Linear ECTR could be used to elongate telomeres by end-joining reactions or by homologous recombination and copy templating. The small size of much of the linear ECTR makes it unlikely that this could account for rapid, large increases in telomere length in ALT cells. ECTR may also be involved in titrating out telomere binding proteins. Although it is entirely possible that the ECTR within APBs is only a subset of the total ECTR in ALT cells, the co-localization within APBs of ECTR and proteins involved in recombination suggests either that they are involved in the ALT mechanism or are its by-products. APBs appear in the late S/G2/M compartment of the cell cycle (Grobelny et al., 2000; Wu et al., 2000), when homologous recombination is most active (Takata et al., 1998).

Proteins that may be involved in the ALT mechanism

Many of the proteins that have been identified in APBs may be involved in the ALT mechanism (Table 2). RAD52, RAD51, RPA, the MRE11/RAD50/NBS1 complex, and RecQ helicases have functions compatible with homologous recombination and recombination-dependent replication. All of these proteins are present in APBs, along with PML protein which is essential for the formation of PNBs. Another common component of PNBs, SUMO-1, is a small ubiquitin-related modifier protein that can be covalently attached to other proteins, including PML, RAD51, RAD52 and PCNA (Lallemand-Breitenbach et al., 2001; Shen et al., 1996; Tanaka et al., 1999; Yeh et al., 2000). Mutation of the S. pombe homologue of SUMO-1 causes a telomere length phenotype (Tanaka et al., 1999).

Other proteins that could conceivably be involved in ALT include poly(ADP-ribose) polymerase (PARP), which binds single and double stranded DNA breaks and poly ADP-ribosylates proteins including itself, causing it to shuttle on and off. It is known to interact with p53 and there is evidence that it is involved in DNA repair and suppressing recombination at double-strand breaks (Tong et al., 2001). In mouse embryo fibroblasts loss of both PARP and p53 results in a telomere length phenotype that somewhat resembles ALT: the mean telomere length is increased by 50% and the standard deviation and range of the length are also increased. In contrast, PARP-/- single mutants have short telomeres and increased chromosomal instability. P53 -/- single null mutants have unchanged average telomere lengths but the variance is increased, although to a lesser extent than the double mutants. The double null mutants have an increased prevalence of tumors compared to single mutants. However, when a tumor in a double null mouse was investigated, it had decreased telomere length (Tong et al., 2001).

It is possible that ALT is controlled by repressors of recombination; these may be specific for the telomere, or may also have a role elsewhere in the genome. The first example is the Rif proteins which may specifically inhibit recombination at the telomeric repeats in S. cerevisiae. Type II telomerase-null survivors are inhibited by Rif2p and to a lesser extent by Rif1p (Teng et al., 2000). The Rif proteins normally interact with the yeast telomere binding protein, Rap1p, and negatively regulate telomere length (Wotton and Shore, 1997). It is postulated that the Rif proteins may interfere with the Rad50p complex. Mammalian homologues of these proteins have not yet been identified. Second, it has been suggested that the mismatch repair genes suppress recombination more generally, especially homeologous recombination, and it has recently been observed that defects in the mismatch repair pathway provide a growth advantage to telomerase-null yeast cells as they approach senescence (Rizki and Lundblad, 2001). As a final example, unlike in yeast cells the telomeres in human cells are partly nucleosomal (Tommerup et al., 1994), so the histone proteins could also be involved in normal suppression of recombination at the telomere. There is evidence in yeast that post-translational modification of histones can regulate recombination (Noma et al., 2001).

Telomeric recombination in normal cells?

Although interest has so far centered on telomere maintenance by recombination in immortalized cells and cancers, there is some evidence in support of normal human cells using a recombination-mediated mechanism to maintain very short telomeres at the expense of longer telomeres. When telomeres on individual chromosome arms in a mass culture of normal human fibroblasts were examined, it was found that the shorter telomeres were maintained above 1 to 2 kb, while the longer telomeres experienced some rapid deletion events (Martens et al., 2000). The proliferation capacity was found to be correlated with the mean telomere length and not the lengths of the four shortest telomeres, supporting the notion that the mechanism is non-reciprocal recombination between long and short telomeres. The authors suggested that the limited reprieve from senescence provided by lengthening the shortest telomeres in this way could be influenced by mutations affecting the cell's predisposition to recombination.

There is some evidence that recombinational telomere lengthening may occur in some mouse cells in vivo under exceptional conditions. In mice that had lost telomerase activity due to a knockout mutation (mTERC-/-), the germinal center lymphocytes lost 7 kb of telomere repeats post immunization, consistent with proliferation in the absence of telomerase. In later generations of mTERC-/- mice there were only a few germinal centers, but the lymphocytes had elongated their telomeres by an average of 12 kb. One possible explanation is the utilization of an ALT-like mechanism (Herrera et al., 2000).

Significance of ALT in human tumors

ALT has been detected in a variety of human tumors as well as tumor cell lines. These include bone and soft tissue sarcomas, glioblastomas, and carcinomas of the lung, kidney, adrenal, breast, and ovary (Bryan et al., 1997a; Mehle et al., 1996; Hakin-Smith et al., submitted; J Henson et al., unpublished). Examples of immortalized human cell lines that have ALT as their only telomere maintenance mechanism are shown in Table 1. All immortalized cell lines studied to date either have telomerase activity or have the telomere length phenotype characteristic of ALT (Colgin and Reddel, 1999), with the possible exception of a lymphocytic cell line that may have both (Strahl and Blackburn, 1996). The situation, however, is more complex for tumors. Approximately 85% of all human tumors have telomerase activity (Shay and Bacchetti, 1997), but an extensive survey to determine the prevalence of ALT in human tumors has not yet been done. It is not possible to conclude that the remaining 15% must by definition use some form of ALT because, as discussed in more detail elsewhere (Reddel, 2000), it is not clear that activation of a telomere maintenance mechanism and immortalization are essential for all tumors. The proportion of ALT+ tumors is further obscured by the occurrence of both ALT and telomerase activity in some tumors (Bryan et al., 1997a). Whether both of these telomere maintenance mechanisms coexist in cells in vivo or just in different subpopulations in the same tumors has not been determined. The latter possibility is supported by the observation that a patient with a telomerase-positive glioblastoma multiforme had an ALT+ recurrent tumor (Hakin-Smith et al., submitted).

ALT may be more common in tumors derived from mesenchymal tissues. This is reflected in the higher prevalence of ALT in immortalized cell lines (~46% ALT, many of which are fibroblastic in origin) compared with tumor-derived cell lines (~5% ALT, mostly carcinomas) (Reddel et al., 2001). Of 210 sarcomas included in six published reports, 56% were telomerase-negative (Aogi et al., 2000; Bovée et al., 2001; Scheel et al., 2001; Schneider-Stock et al., 1999; Yan et al., 1999; Yoo and Robinson, 2000). Mesenchymal compartments mostly have slower cell turnover and less telomere shortening than in many epithelia, and may therefore repress telomerase more tightly. Even if the probability of ALT being activated is the same during the genesis of carcinomas and sarcomas, tighter repression of telomerase in mesenchymal cells may mean that the relative probability of activating ALT is higher in sarcomas than in carcinomas. Although the overall numbers are small, ALT seems to occur frequently in Li-Fraumeni syndrome (LFS) immortal cell lines (Table 1) and tumors (Bryan et al., 1997a). The reason for this is unclear and may relate to loss of p53 being an early event in the genesis of LFS tumors; p53 interacts with RAD51 (Buchhop et al., 1997) and loss of p53 increases homologous recombination (Bertrand et al., 1997). It is interesting to note that sarcomas are a feature of the tumor spectrum in LFS. For reasons which are also unclear, there are a few types of carcinomas that appear to have a relatively low incidence of telomerase positivity. For example, of a total of 237 papillary thyroid carcinomas described in 12 reports 53% did not have detectable telomerase activity (Aogi et al., 1998, 1999; Brousset et al., 1997; Cheng et al., 1998; Haugen et al., 1997; Kammori et al., 2000; Lo et al., 1999; Matthews et al., 2001; Okayasu et al., 1997; Saji et al., 1997, 1999; Yashima et al., 1997). How many of these have ALT is currently unknown.

ALT may be relevant for diagnosis, prognosis, and treatment of cancer. A number of studies have attempted to use the presence of telomerase activity to distinguish benign from malignant tumors (Hiraga et al., 1998; Kammori et al., 2000; Matthews et al., 2001; Saji et al., 1997; Yashima et al., 1997). It is possible that the correlations would be improved if ALT were also taken into account. The type of telomere maintenance mechanism used by tumors may have prognostic significance. For example, patients with ALT+ high grade glioblastomas have a significantly longer survival than those that are ALT-negative (Hakin-Smith et al., submitted). It may therefore be appropriate to stratify management protocols for some tumor types according to telomere maintenance mechanism. Regarding treatment, an implication of the existence of ALT is that tumors using this telomere maintenance mechanism (including mixed telomerase-positive/ALT+tumors), will be resistant to telomerase inhibitors. Also, telomerase inhibitors will put tumors that are initially telomerase-positive under strong selection pressure for activation of ALT. Repression of ALT in ALT+ immortalized cell lines results in senescence and cell death (Nakabayashi et al., 1997; Perrem et al., 1999), so ALT, like telomerase, may be an attractive drug target. Combination therapy using ALT and telomerase inhibitors may help prevent the emergence of drug resistance.

Acknowledgements

The authors thank Clare Fasching for comments on the manuscript. Work in the authors' laboratory is supported by Cancer Council NSW and the National Health and Medical Research Council of Australia.

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Figures

Figure 1 Terminal restriction fragment (TRF) length analysis of IIICF/a2 cells, showing temporal correlation between immortalization and occurrence of the telomere length pattern characteristic of ALT. This culture entered crisis at population doubling (PD) 76, and by PD 77 a few weeks later immortalized cells had overgrown the culture; telomerase activity was not detectable at any time (Yeager et al., 1999). TRF analysis was done by digesting genomic DNA with restriction enzymes that do not recognize the telomeric sequence, TTAGGG, separating it by pulsed field gel electrophoresis, and then hybridizing the dried gel with a radioactively labeled probe complementary to the (TTAGGG)n sequence. Reproduced from (Yeager et al., 1999) with permission of the publisher

Figure 2 Visualization of telomeres in (a) ALT and (b) telomerase-positive cells by fluorescence in situ hybridization (FISH), showing the heterogeneous telomere lengths in ALT cells. A fluorescently labeled probe for the telomeric DNA sequence was hybridized to metaphase spreads

Figure 3 ALT-associated PML bodies (APBs) in a human ALT cell line (a-c) and a telomerase negative human sarcoma (d). Immunohistochemistry was performed using PML (b,d) and TRF1 (a) or TRF2 (d) antibodies. PML and the TRF proteins were visualized with Texas Red- and FITC-conjugated secondary antibodies, respectively. Nuclei in the sarcoma were counterstained with DAPI (d). Panel c is the merge of a and b, and shows the colocalization of TRF1 and PML in nuclear aggregates. Colocalization of TRF2 and PML in the sarcoma is indicated by arrowheads (d)

Figure 4 FISH with subtelomeric CEPH mega-YAC probes specific for (a) chromosome 13 and (b) chromosome 14 on metaphases of an ALT cell line. No subtelomeric translocation events can be detected, consistent with the telomeric recombination events being specific (Dunham et al., 2000) rather than reflecting a generalized increase in recombination frequency in ALT cells. Probes were kindly provided by Dr Thomas Haaf, Max-Planck-Institute of Molecular Genetics, Berlin, Germany

Figure 5 Homologous recombination dependent replication of telomeres. Four proposed mechanisms, described in more detail in the text, are represented by the DNA structures involved. These structures arise when the 3' telomeric end invades an homologous telomeric repeat array forming a D-loop. These structures look identical locally (within the dashed rectangle), and also resemble a replication fork. Similar mechanisms involved in DNA replication may allow extension of the 3' invading strand. Lagging strand synthesis may then be templated on the D-loop with a cross over event(s), or templated on the newly synthesized 3' strand with branch migration

Tables

Table 1 Examples of human ALT cell lines

Table 2 Proteins found in ALT-associated PML bodies (APBs)a

21 January 2002, Volume 21, Number 4, Pages 598-610
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